1. Technical Field
The present disclosure relates to a confocal optical detector, to a detector array, and to a manufacturing method thereof.
2. Description of the Related Art
As is known, today available are light detectors, even miniaturized ones, as well as microanalyzers, the latter being also known as “micro-scanners”.
In general, light detectors enable scanning of small portions of specimens to be analyzed in order to determine characteristics and/or properties of the specimens. For the above purpose, light detectors, also known as “optical detectors”, usually comprise optical elements and movement devices having particularly small dimensions, for example of the order of some millimeters. In addition, optical detectors have been proposed, in which the movement devices are formed by microelectromechanical systems (MEMS).
In general, optical detectors envisage illuminating with a first light beam a portion of a specimen, and then receiving and analyzing a second light beam coming from the specimen itself, whether generated by reflection of the first light beam by the specimen or else by excitation of the specimen following upon the incidence of the first light beam. In the latter case, they are commonly referred to as optical detectors based upon the phenomenon of light-induced fluorescence, or else, more briefly, as “fluorescence detectors”.
Irrespective of the details of construction, optical detectors find wide use in sectors such as, for example, diagnostics for purposes of medical research. For example, in diagnostic field it is known to couple optical detectors to diagnostic devices.
In general, the diagnostic devices each include a respective assay. In turn, the assay may comprise a solid substrate, which is typically of a flat type and has a surface that is functionalized so as to present detection areas, within which receptors provided with specific markers, described hereinafter, are immobilized.
In practice, by “receptor” is meant any member of a pair or of an n-tuple of elements that can bind together. Consequently, each receptor is able to couple, or in any case react, with a respective binding mate, or else with a respective plurality of binding mates, enabling detection thereof. For example, the receptors may comprise biomolecules (DNA, RNA, proteins, antigens, antibodies, aptenes, sugars, etc.) or chemical species, or micro-organisms or parts of them (bacteria, viruses, spores, cells, etc.).
As regards the markers, each of them is such that, when the corresponding receptor couples or interacts with its own binding mate, or else with its own binding mates, it is activated. In particular, in the so-called fluorescence diagnostic devices, if an activated marker is excited with a light radiation at a certain wavelength λe, it emits a light radiation of its own having a wavelength λf different from the wavelength λe. In general, these markers are known as “fluorescence markers”.
By way of example, known to the art are three-component binding assays, which use, each, a first immobilization of a first antibody to a solid substrate, this first antibody being able to couple with an antigen present in a specimen solution. Coupling with the antigen is then detected thanks to a second antibody, which functions as the marker and couples with a different epitope of the same antigen. The second antibody has a fluorescent label attached thereto; consequently, the amount of fluorescence is correlated to the amount of antigens present in the specimen solution.
In practice, by detecting, by means of an appropriate optical detector, the light radiation at the wavelength λf, it is possible to derive information on the chemico-physical characteristics of the specimen to be analyzed, since the light intensity detected is a function of the amount of markers activated in the assay, and hence of the amount of molecules or biomolecules detected from the assay. For the above purpose, the optical detector must be sensitive to the wavelength λf of the light radiation emitted by the markers.
This being said, optical detectors are known that are particularly suited for detection of the electromagnetic radiation emitted by markers, especially by fluorescent markers.
In particular, known to the art are the so-called “confocal detectors”, as described, for example, in U.S. Pat. No. 3,013,467 and a principle diagram of which is shown in FIG. 1.
In detail, a confocal detector 1, also known as “confocal microscope”, is formed by a laser source 2, by an optoelectronic sensor 4, by an optical beam splitter 6, by a first lens 8, by a so-called “pinhole” 10, and by a second lens 12. FIG. 1 moreover shows an element to be analyzed S, which may be formed by an assay on which a specimen to be analyzed has been laid.
In greater detail, the pinhole 10 and the first and second lenses 8, 12 are optically aligned; i.e., they have substantially coinciding optical axes, which hence define a system axis OA. In practice, assuming for simplicity that the first and second lenses 8, 12 are thin and have, respectively, a first optical center O1 and a second optical center O2, the first and second optical centers O1, O2 lie along the system axis OA. In addition, P is the center of the pinhole 10, which also lies along the system axis OA. Furthermore, the pinhole 10 is arranged between the first lens 8 and the second lens 12.
The optoelectronic sensor 4 is usually aligned with respect to the system axis OA. Moreover, the optical beam splitter 6 also intercepts the system axis OA and is arranged between the optoelectronic sensor 4 and the pinhole 10.
As regards, instead, the laser source 2 and the element to be analyzed S, the laser source 2 is arranged laterally with respect to the system axis OA, whilst the element to be analyzed S intercepts the system axis OA, with respect to which it is substantially aligned.
In greater detail, the laser source 2 and the optical beam splitter 6 are arranged in such a way that, if we refer to the “first optical beam F1” to indicate the electromagnetic radiation emitted of the laser source 2, the first optical beam F1 propagates from the laser source 2 in a first direction of propagation D, until it impinges upon the optical beam splitter 6, which reflects a first portion thereof in the direction of the first lens 8, along the system axis OA. In particular, the first direction of propagation D forms an angle of 90° with the system axis OA. Hence, if the portion reflected by the optical beam splitter 6 is once again referred to as “first optical beam F1” (i.e., if we neglect the portion of first optical beam F1 that is not reflected by the optical beam splitter 6), the first optical beam F1 follows an optical path that forms an angle of 90°.
After is has been reflected by the optical beam splitter 6, the first optical beam F1 is focused by the first lens 8 at the center P of the pinhole 10, and then propagates until it impinges on the second lens 12, which focuses it on an image point A, which is arranged at the intersection of the system axis OA with an image plane PF of the second lens 12 itself.
In practice, in order to get the first optical beam F1 to follow the path described, and assuming for simplicity that the first and second lenses 8, 12, in addition to being thin, are biconvex, the center P of the pinhole 10 and the image point A are conjugate points; i.e., if we assume setting, in absence of the element to be analyzed S, a pointlike object in the image point A, it forms a corresponding image at the center P of the pinhole 10, and moreover, if we assume setting this pointlike object at the center P of the pinhole 10, it forms a corresponding image in the image point A. Once again in other words, if PP is the plane of the pinhole 10, orthogonal to the system axis OA and containing the center P, the plane PP of the pinhole 10 and the image plane PF of the second lens 12 are conjugate planes of the second lens 12. This explains why the confocal detector 1 is referred to as “confocal”.
Operatively, in the case where in the image point A an activated marker is present, when it is illuminated by the first optical beam F1, it generates a second optical beam F2; the first and second optical beams F1, F2 may have, respectively, the wavelength λe and the wavelength λf.
The second optical beam F2 propagates from the image point A up to the second lens 12, by which it is focused at the center P of the pinhole 10. Next, the second optical beam F2 propagates through the first lens 8 and the optical beam splitter 6, until it impinges on the optoelectronic sensor 4. In particular, as regards the optical beam splitter 6, it exhibits a dichroic behavior, i.e., albeit reflecting at least in part radiation at the wavelength λe, is transparent for radiation having wavelength λf; hence, it does not interfere with the second optical beam F2.
The optoelectronic sensor 4 is hence able to detect and process the second optical beam F2, on the basis of which it determines chemico-physical characteristics of the element to be analyzed S. In particular, thanks to the presence of the pinhole 10, on the optoelectronic sensor 4 there impinge, to a first approximation, only optical rays that, in addition to forming the second optical beam F2, are originated exactly from the portion of the element to be analyzed S present in the image point A. In fact, any possible other optical rays (designated by F3 in FIG. 1) generated by portions of the element to be analyzed S that are arranged in points different from the image point A, are filtered by the pinhole 10 before reaching the first lens 8, and hence do not reach the optoelectronic sensor 4. This prevents formation of so-called “artifacts”, i.e., spurious light signals generated by points other than the image point A, for example points arranged at the intersection of the system axis OA with planes parallel to the image plane PF of the second lens 12, which could cause a deterioration of the performance of the confocal detector 1.
If the confocal detector 1 is equipped with an appropriate device (not shown) for movement of the element to be analyzed S, it hence makes it possible to obtain three-dimensional images of the element to be analyzed S, enabling a fast and effective analysis of the specimen to be analyzed S itself. Alternatively, and once again for this purpose, the confocal detector 1 may be equipped with a system for movement of at least one component thereof, such as, for example, the optical beam splitter 6.
Even though the confocal detector 1 is hence suited to the analysis of specimens, on account of the optical paths followed by the first and second optical beams F1 and F2 it is not easy to use in the case where a detector array is to be provided, i.e., a matrix of confocal detectors coplanar to one another. In fact, the overall dimensions of the confocal detector 1 may prove excessive for this kind of applications.